The freezing and thawing of bulk protein solutions maximizes production capacity while enabling drug product logistics to be better aligned with market demands. Freezing also eliminates risks from stress during transport and minimizes the possibility of microbial growth. However, the potential for freezing-induced aggregation, caused by cryoconcentration, crystallization of solutes or buffer salts, pH changes, and ice water interface-induced denaturation, results in significant challenges for success with this unit operation. From a processing standpoint, freezing and thawing rates measured on the basis of the time required to complete phase transition and storage temperature in relation to the glass transition temperature are critical parameters. This two-part article focuses on freezing protein solutions with an active freeze–thaw system called the cryovessel. Part 1 of this article presents results from a detailed mapping of solute distribution and solution property changes during the freezing stage for various processing rates as a function of position in the cryowedge. Part 2 of this article will present results from examination of the frozen state itself. Data has been generated on a scaled-down model, called the cryowedge, to simulate the freezing and thawing process in a cryovessel. The results illustrate the extent of changes in various solution properties that occur during freezing at different rates.

(PFIZER, INC.)

Freezing and thawing large volumes of protein bulk drug substance (BDS) has become an important step in the manufacture of biotherapeutics. Producing biologics is an expensive process and to optimize capacity use, the BDS is often produced in large manufacturing campaigns. The bulk solution is converted into drug product based on market demand, and therefore may have to be stored for relatively long periods of time. To decouple the production of the bulk solution from that of the drug product, the bulk is often stored in the frozen state. Other advantages of this storage option include slower chemical degradation kinetics, minimizing microbial growth and the eliminating transportation stress by reducing air–water interfaces.1-3 A successful bulk storage program enhances bioprocess capacity use and reduces the overall cost of production.

Despite the advantages of freezing BDS, the freeze–thaw process itself offers numerous challenges because of the complexity of the changes that occur and the poor understanding of how the solutes, including the protein, are affected. Although a number of studies have been published on the impact of freezing proteins, these are limited to small or microscopic volumes where process conditions become irrelevant compared to practical BDS freezing systems. In studies where the rate is taken as a study parameter, this mostly refers to the rate of cooling (i.e., the rate at which the temperature is lowered down to the freezing point).4 However, the more critical parameter is the time taken to actually freeze the solution (the phase transition time), because it is during this stage that cryoconcentration, solute redistribution, phase separation, ice-interface creation, eutectic crystallization, glass formation, pH change, and other freezing-related changes occur. A number of these phenomena can be destabilizing to the protein structure as it cryoconcentrates along with the other solutes during this phase transition freezing stage before complete solidification. Protein molecules that have become (partially or fully) unfolded during the freezing stage potentially can lead to aggregate formation over time because these unfolded molecules serve as nuclei and interact with their neighbors if enough mobility is afforded to the system by storage above or near the glass transition temperature (Tg'). The outcome of the process in terms of the stability of protein on frozen storage is likely to be significantly determined by what occurs during the freezing stage.1,2

Time required to freeze the solution (i.e., the time to complete the phase transition) is dependent on the volume or mass to be frozen and the rate of heat removal. Literature studies using slow or fast rates mainly have used small volumes where the time required for freezing, even when described as slow, is much shorter than for practical systems involving the processing of tens to hundreds of liters of solution. Furthermore, when the heat and mass transfer dimensions are small, significant solute movement and cryoconcentration effects are not in place before immobilization traps the solutes. Thus, while fundamental phenomena such as ice-interface effects, phase separation, eutectic crystallization, and pH change can be explored in small-volume studies, the effect of cryoconcentration and solute redistribution, or time in phase transition, require studies to be conducted at scales or dimensions more representative of the full-scale process. A fundamental mechanistic understanding of the behavior of a representative-scale system undergoing freeze–thaw processing will enable better definition of the process parameters to minimize the impact of processing on protein drug substance.

Several options are used in the biotech industry for freezing biologics. These include passive systems such as bottles or carboys, and active systems such as tanks with coolant circulation (such as CryoVessels, Sartorius-Stedim Biotech, Aubagne, France; and FreezeContainer, Zeta Holdings, Styria, Austria) and large-scale bag freezing systems (Celsius, Sartorius-Stedim). Various studies have been published on solute distribution in such freezing systems, although primarily in frozen state.5–9 However, a thorough study of the evolution of solute distribution during the freezing process, especially in a cryovessel, is not available. Additionally, the connection between concentration changes during freezing and cryoconcentration in the frozen state has not been explored. It has been shown that cryoconcentration in the frozen state is maintained to a great extent if thawing is performed in a static manner.10,11 In this paper, we have focused on mapping cryoconcentration behavior in large scale systems. Through a series of experiments, we have investigated the solute distribution pattern in a Cryowedge (Sartorius-Stedim) when a protein solution is subjected to various freezing cycles. The cryowedge geometry represents a scaled-down model for commercially available controlled rate freeze–thaw systems called CryoVessels. The heat and mass transfer surfaces or distances in the wedge are identical to those in a cryovessel of corresponding size, so that the freeze and thaw behavior of a full-scale vessel can be studied at a laboratory scale. The study was conducted on a Cryowedge 34, which is representative of a 300-L cryovessel. This article (Part 1) presents results from a detailed mapping of solute distribution and solution property changes during the freezing stage at various processing rates as a function of position in the cryowedge. Part 2 of this article will present results from an examination of the frozen state itself.